Name: a possible zebrafish model of polycystic kidney disease: knockdown of wnt5a causes cysts in zebrafish kidneys
Uploaded: 2014-12-02T11:00:00.000+00:00
Duration: 10 min 51 s
Description: Watch this Scientific Journal Video about A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys at JoVE.com

这项工作是支持由美国国立卫生研究院（DK093625到LH和DK069909和DK047757至JHL）和VA（优异奖I01BX000820到JHL）。我们要感谢博士迈克尔·包和何洁博士在宾夕法尼亚斑马鱼核心的大学提供必要的支持。

注:伦理学声明:所有斑马鱼实验批准的机构动物护理和使用委员会在东弗吉尼亚医学院。 1.准备吗啉<ol><li>设计和合成的翻译阻滞（AUG-）和剪接抑制（剪接）反义吗啉代（MO）的寡核苷酸用于感兴趣按照制造商的说明（ 图1A）的基因。请参阅生产商的表1中的信息。 注:材料订单出货冻干股玻璃瓶。 </li><li>添加高档无菌水至玻璃瓶重新暂停的MO至25微克/微升的最终浓度。确保该寡核苷酸被完全溶解。如果一些固体遗骸，加热包含在65℃的股票的寡核苷酸溶液中5至10分钟，涡旋简要小瓶。 </li><li>存储在MO原液在RT。不要将它们存储在4℃或-20℃; C，因为较低的温度可能导致在MO寡核苷酸结合到容器壁。测量使用分光光度计原液的浓度（请参阅表1）一个新的MO原液制成各一次。 </li><li>通过稀释原液用高档无菌水至所需的剂量准备注射的当天将MO工作溶液。加0.5％酚红达到0.05％的浓度。例如，以与15毫微克/ nL的浓度5μl的工作溶液中，加入3微升的MO原液和0.5微升0.5％酚红至1.5微升水。 注:有了这个工作浓度，注射每一滴（500 PL）包含MO的7.5纳克和两滴含有15纳克MO的。 </li></ol> 2.准备的注射装置的<ol><li>购买玻璃预拉针（请见表1详细信息）。另外，拉玻璃注射针无线TH针拉马。 </li><li>将空气压缩机和调整压力设置为50磅。打开解剖显微镜光源和微微微量注射泵和调整设置如下:保持压力20磅，喷射压力10磅，2.5和100微秒范围选通的周期值。 </li><li>加载玻璃预拉针以5微升注射溶液和放置针在与尖端朝下的垂直位置。等待，直到所有的溶液到达针尖没有可见气泡。将针保持器在旁边的显微镜适当的位置，并插入玻璃针插入支架。调整喷射角为45度。 </li><li>把针尖进入视野，在显微镜下，高走下舞台，并专注于尖端最薄的区域。断针尖与细点镊子。 </li><li>将尺子和毛细管具有0.15mm的一侧通过显微镜下侧内径;该溶液注入到毛细管的一端，以使一个液柱。调整排出时间来达到每17滴每1mm长度的液柱，然后每滴的体积为1 NL（墨滴体积液柱=πX半径2×长度（）滴/计数（）。 注:将一滴用直径为0.1毫米给出约500复一个注射量（墨滴体积= 4/3 XπX半径3）。 </li></ol> 3.准备施肥斑马鱼胚胎和注射用吗啉代<ol><li>根据标准斑马鱼的饲养和维护协议公布转基因斑马鱼繁殖对:设置的Tg（GFP wt1b）。收集自然产卵后的胚胎，并将其保存在一个10cm培养皿充满E3水（5毫米氯化钠，氯化钾0.17毫米，0.33毫米氯化钙2，0.33毫米硫酸镁4）。启动MO注射马上13。监测胚胎形态，在显微镜下，并确保MO注射是在单细胞阶段，或不迟于四细胞阶段。 <ol><li>胚胎转移到10cm的培养皿中以楔形琼脂槽。吸出多余的水E3并轻轻按压胚胎进入低谷。 </li><li>操纵与微量的胚胎以可视化的解剖显微镜下1-细胞期的胚胎。穿透绒毛膜，然后将蛋黄到一个或两滴MO的注入蛋黄（1滴= 500 PL）。转移注射的胚胎至10cm的培养皿与E3的水和孵化在28.5℃。 </li></ol></li><li>在注射后，取出死胚，并记录注射的胚胎的数量。取代E3水在盘每24至48小时。 </li></ol> 4.表型救援实验<ol><li>选择来自另一个物种具有不同的主碱基对结构（通常为3-7个碱基对的错配），并且，因此，耐对MO的直向同源物，救援。例如，在这个实验中，我们选择了鼠标因为鼠标Wnt5a的 mRNA序列是来自斑马鱼的序列不同。 </li><li>设计的引物组与正向引物起始于平移位点和反向引物在接近所述mRNA编码区的末端。添加T7启动子序列的正向引物序列的5&#39;端。在这个实验中，我们使用了以下的引物序列;前锋:5&#39;-TAA TAC GAC TCA CTA标签GGA CTA TGA TGC TGC TGA AGC TGA A- 3&#39;和反向:5&#39;-TCA CTT GCA GAC GTA CTG gtc- 3"。 </li><li>执行一个50μl的聚合酶链反应（PCR）用引物组（0.5μM，每种引物）上述设计和0.1微克含有小鼠Wnt5a的 cDNA序列与下列PCR程序模板DNA:1个循环的95℃5分钟; 35个循环的95℃（30秒），58.5℃（30秒），72℃（1分钟）;和最后延伸步骤在72℃进行7分钟。 </li><li>完成后，吨他的周期，运行2微升的PCR产物的1％琼脂糖凝胶，以确保乐队是正确的大小。纯化的PCR产物与根据制造商的说明一PCR纯化试剂盒。检查DNA浓度用分光光度计。存储所述纯化的PCR产物，在-20℃或在加帽的RNA转录在下一步直接使用。 </li><li>合成体外根据制造商的说明书加帽的mRNA用加帽的RNA合成试剂盒。稀释的mRNA在20μl无RNA酶的水，并确定该浓度。分装的RNA，并储存于-80℃。 </li><li>对注射的当天，制备mRNA的核糖核酸酶与游离的无菌水的工作溶液。需要注意的是40皮克通常是在该RNA的非特异性或毒性作用不发生的最高的RNA剂量。置于冰上的工作方案。注入胚胎与MO，然后抢救的mRNA，或二者共同注入在一起。例如，如果与mRNA prepar的浓度每次注射的ED在步骤4.5为0.6微克/微升，添加0.67微升的mRNA（0.4微克）与4.33微升无RNA酶的水，使0.08微克/微升的最终浓度，然后一滴（500 PL）中含有40皮克mRNA的。准备MO如步骤3中所述。 </li></ol> 5.准备注射胚胎的荧光成像<ol><li>孵育28.5°CO后/ N，增加0.003％N-苯基硫氧嘧啶（PTU）的E3水，以防止黑化，从而影响观察用荧光显微镜前肾结构。然而，原肠胚形成之前不要添加PTU，因为它会影响早期胚胎发育。 </li><li>在48 HPF，手动dechorionate用细镊子胚胎。采取48和72 HPF胚胎图像光解剖显微镜下。 注意:这些图片可以采取无需麻醉。 </li><li>约10分钟前在荧光显微镜下成像，将它们放置在麻醉胚胎10cm的培养皿含有160微克/毫升缓冲三卡因。等待5-10分钟，然后轻轻地接触斑马鱼，以确认它们不移动，因此，麻醉正在工作。 </li><li>之后将胚麻醉，将它们安装在3％的甲基纤维素和定向它们在解剖显微镜下俯卧位置（请参阅表1）。 </li><li>观察在荧光显微镜下的前肾结构（请参阅表1），并拍摄照片，在不同的放大倍数（10X和20X）。 </li></ol>

<ol>
	<li>Choi, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdc42+deficiency+causes+ciliary+abnormalities+and+cystic+kidneys.">Cdc42 deficiency causes ciliary abnormalities and cystic kidneys.</a> J Am Soc Nephrol. 24 (9), 1435-1450 (2013).</li><li>Hostetter, C. L., Sullivan-Brown, J. L., Burdine, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+pronephros:+a+model+for+understanding+cystic+kidney+disease.">Zebrafish pronephros: a model for understanding cystic kidney disease.</a> Dev Dyn. 228 (3), 514-522 (2003).</li><li>Nyengaard, J. R., Bendtsen, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glomerular+number+and+size+in+relation+to+age,+kidney+weight,+and+body+surface+in+normal+man.">Glomerular number and size in relation to age, kidney weight, and body surface in normal man.</a> Anat Rec. 232 (2), 514-201 (1992).</li><li>Keller, G., Zimmer, G., Mall, G., Ritz, E., Amann, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nephron+number+in+patients+with+primary+hypertension.">Nephron number in patients with primary hypertension.</a> N Engl J Med. 348 (2), 101-108 (2003).</li><li>Cullen-McEwen, L. A., Kett, M. M., Dowling, J., Anderson, W. P., Bertram, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nephron+number,+renal+function,+and+arterial+pressure+in+aged+GDNF+heterozygous+mice.">Nephron number, renal function, and arterial pressure in aged GDNF heterozygous mice.</a> Hypertension. 41 (2), 335-340 (2003).</li><li>Gerlach, G. F., Wingert, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kidney+organogenesis+in+the+zebrafish:+insights+into+vertebrate+nephrogenesis+and+regeneration.">Kidney organogenesis in the zebrafish: insights into vertebrate nephrogenesis and regeneration.</a> Wiley Interdiscip Rev Dev Biol. 2 (5), 559-585 (2013).</li><li>Igarashi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview:+nonmammalian+organisms+for+studies+of+kidney+development+and+disease.">Overview: nonmammalian organisms for studies of kidney development and disease.</a> J Am Soc Nephrol. 16 (2), 296-298 (2005).</li><li>Zhou, W., Boucher, R. C., Bollig, F., Englert, C., Hildebrandt, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+mesonephric+development+and+regeneration+using+transgenic+zebrafish.">Characterization of mesonephric development and regeneration using transgenic zebrafish.</a> Am J Physiol Renal Physiol. 299 (5), 1040-1047 (2010).</li><li>Liu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+defect+in+a+novel+Nek-family+kinase+causes+cystic+kidney+disease+in+the+mouse+and+in+zebrafish.">A defect in a novel Nek-family kinase causes cystic kidney disease in the mouse and in zebrafish.</a> Development. 129 (24), 5839-5846 (2002).</li><li>Slanchev, K., Putz, M., Schmitt, A., Kramer-Zucker, A., Walz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nephrocystin-4+is+required+for+pronephric+duct-dependent+cloaca+formation+in+zebrafish.">Nephrocystin-4 is required for pronephric duct-dependent cloaca formation in zebrafish.</a> Hum Mol Genet. 20 (16), 3119-3128 (2011).</li><li>Huang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+Wnt5a+in+prostate+gland+development.">The role of Wnt5a in prostate gland development.</a> Dev Biol. 328 (2), 188-199 (2009).</li><li>Andre, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Wnt+coreceptor+Ryk+regulates+Wnt/planar+cell+polarity+by+modulating+the+degradation+of+the+core+planar+cell+polarity+component+Vangl2.">The Wnt coreceptor Ryk regulates Wnt/planar cell polarity by modulating the degradation of the core planar cell polarity component Vangl2.</a> J Biol Chem. 287 (53), 44518-44525 (2012).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev Dyn. 203 (3), 253-310 (1995).</li><li>Torres, V. E., Harris, P. C., Pirson, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autosomal+dominant+polycystic+kidney+disease.">Autosomal dominant polycystic kidney disease.</a> Lancet. 369 (9569), 1287-1301 (2007).</li></ol>

The authors have nothing to disclose.

多囊肾（PKD）是终末期肾脏疾病的主要原因在人类中之一，其特征在于进行性囊肿形成，肾肿大，与异常小管发育14。常染色体显性多囊肾（ADPKD）是一种遗传性疾病，其中突变要么PKD1的，编码多囊蛋白1（PC1），或PKD2，多囊编码-2（PC2），导致多囊性肾脏。许多其它基因，特别是那些编码在初级纤毛中发现的蛋白质，被认为是参与肾囊肿的发展，至今尚无理想的工具来筛选这些基因。我?...

斑马鱼（ 斑马鱼 ）胚胎已被广泛用作模型为研究肾发展和多囊肾病。有许多优势，使用斑马鱼作为动物模型:研究遗传相互作用的可行性，使用反义吗啉（MO），用于蛋白质敲低的能力，有机会迅速测定大量的胚胎，并且容易观察器官的表型活幼虫1。该前肾是第一个肾在脊椎动物发展和功能是在幼虫斑马鱼2。斑马鱼前肾的结构相对简单相比哺乳动物后肾，第三和最后肾发展的哺乳动物。肾单位是肾脏的工作单元，其中每个人肾脏500,000-1,000,000肾3,4和具有约13000肾5各小鼠肾脏之间含有，因此很难观察单肾结构我ñ人或小鼠肾脏。斑马鱼只有两个肾单位，每个斑马鱼肾包含所有在肾小球和小鼠和人类6和类似专门肾细胞类型的小管中发现的主要组件。相比其他脊椎动物模型如爪蟾 ，斑马鱼肾更接近于哺乳动物肾，因为它有一个封闭的系统7。 在最近几年中，斑马鱼的基因组已经被测序，允许广泛介绍的遗传工具，广泛突变体资源，并在斑马鱼的模型转基因报告线路集合。 12-72小时受精后（HPF）之间的斑马鱼前肾表格可在透明的胚胎很容易被可视化。在肾母细胞瘤蛋白WT1是肾脏发育的一个重要因素。所述wt1b启动子的Tg的控制下表达绿色荧光蛋白（GFP）转基因斑马鱼线（wt1b:GFP）显示GFP expre裂变专门设在pronephric地区的斑马鱼胚胎，从17 HPF 8起。 Nephronophthisis（NPHP），一种常染色体隐性囊性肾脏疾病，是由NPHP基因9的突变引起。 NPHP4被击倒吗啉导致囊肿形成的的Tg（wt1b:GFP）鱼10因此，这种转基因鱼是一个合适的模型，观察肾脏结构和囊肿形成肾脏发育过程中。重要的是，肾脏发育的调节剂的影响，可以利用该菌株中一个时间和劳动高效方式进行研究。 我们的论文描述了使用的Tg（wt1b:GFP）鱼作为模式基因调制后显现肾囊肿形成。我们使用的开始和剪切位点反义的MO打掉了Wnt5a基因斑马鱼。而Wnt5a是Wnt信号家族的非规范的分泌糖蛋白，发挥各器官和细胞产后的发展具有重要作用功能11。 Wnt5a的工作原理是通过非经典Wnt途径，包括平面细胞极性（PCP）途径，已经发现肾小管伸长时发挥导向细胞分裂的作用。 Wnt5a的通过形成与受体样酪氨酸激酶（RYK），其中，还转导Wnt5a的信令通过形成与VANGL平面细胞极性蛋白2（Vangl2）的复合物，从而促进Vangl2稳定性12复杂调节的Wnt / PCP通路。在PCP途径缺陷可能导致随机的细胞分裂，并导致肾囊肿形成。我们使用的Tg（wt1b:GFP）斑马鱼线以下的Wnt5a敲除，观察肾囊肿形成。 的Tg（wt1b:GFP）斑马鱼模型允许实时成像，及时观察肾脏结构。 Wnt5a的击倒后，肾囊肿形成被发现开始，在24 HPF;在72 HPF，囊肿可能在肾小球和近端小管中找到。这种方法也可用于为screen其他基因可能导致肾囊肿形成。

<table><tbody><tr><td>0.5% Phenol-Red</td><td>Sigma</td><td>P0290</td><td>Color indicator for injection</td></tr><tr><td>Morpholino</td><td>Genn Tools, LLC</td><td>(customized)</td><td>Customized designed to the gene of interest</td></tr><tr><td>Pre-pulled needle</td><td>Tritech Research</td><td>MINJ-PP</td><td>If large amount of needle is required, you can also purchase a needle puller and prepare the needle in the lab</td></tr><tr><td>T7 mMessage Kit</td><td>Ambion</td><td>1344</td><td>For in vitro transcription to make capped mRNA for rescue experiment</td></tr><tr><td>N-Phenylthiourea</td><td>Sigma</td><td>P7629</td><td>For prevention of melanization</td></tr><tr><td>Tricaine</td><td>Sigma</td><td>A5040</td><td>Also called ethyl 3-aminobenzoate, for zebrafish anesthesia</td></tr><tr><td>Methyl Cellulose</td><td>Sigma</td><td>M-0387</td><td>For position of zebrafish&amp;nbsp;</td></tr><tr><td>QIAquick PCR Purification Kit&amp;nbsp;</td><td>Qiagen</td><td>28104</td><td>For purification of PCR product for cap RNA synthesis.</td></tr><tr><td>dNTP mix</td><td>Promega</td><td>U1511</td><td>For PCR</td></tr><tr><td>Tag DNA Polymerase</td><td>Invitrogen</td><td>10342-053</td><td>For PCR</td></tr><tr><td>NanoDrop spectrophotometer</td><td>Thermo Scientific</td><td>ND-1000</td><td>For measure morpholino and RNA concentration</td></tr><tr><td>Air Compressor</td><td>Werther International, Inc.</td><td>Panther Compact 106</td><td>Air source for injection</td></tr><tr><td>Pico micro-injection pump</td><td>World Precision Instruments Inc</td><td>PV830 Pnematic PicoPump</td><td>Other types of microinjection system can be used.</td></tr><tr><td>Micro-manuplator</td><td>World Precision Instruments Inc</td><td>MMJR</td><td>Right-handed (MMJL for left handed)</td></tr><tr><td>Needle holder</td><td>World Precision Instruments Inc</td><td>5430-ALL</td><td>To hold needle for micromanipulation</td></tr><tr><td>Dumont Tweezers</td><td>Fine Surgical Tools</td><td>11253-20</td><td>For breaking off the needle tip</td></tr><tr><td>Dissecting microscope</td><td>Leica M205C&amp;nbsp;</td><td></td><td>For observing and imaging zebrafish embryos</td></tr><tr><td>Fluorscence microscope</td><td>Zeiss Axio Obserer D1m &amp;nbsp;</td><td></td><td>For imaging zebrafish pronephros</td></tr><tr><td>Capillary tube (I.D. 0.15 mm)</td><td>VitroCom</td><td>CV1525Q-100</td><td>For measure the volume of each injected drop</td></tr></tbody></table>

a possible zebrafish model of polycystic kidney disease: knockdown of wnt5a causes cysts in zebrafish kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to &#8220;off-target&#8221; effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

Wnt5a的击倒被引进翻译阻挡MO实现（AUG-MO）或外显子/内含子的剪接边界MO（拼接-MO），以斑马鱼胚胎的一个细胞阶段。所述AUG-MO靶向起始密码子，因此，抑制了母亲和合子Wnt5a的消息。拼接-MO靶向第三剪接供体位点和抑制Wnt5a的（ 图1A）仅合子转录。所述AUG-和剪接morphants phenocopied彼此具有多个缺陷，包括卷曲的尾巴向下体轴和心包水肿（ 图1B）。鼠标Wnt5a的mRN...

Watch this Scientific Journal Video about A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys at JoVE.com

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

多囊肾一种可能的斑马鱼型号:击倒<em&gt; Wnt5a的</em&gt;原因在斑马鱼肾囊肿

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

a-possible-zebrafish-model-polycystic-kidney-disease-knockdown-wnt5a

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

11.1K 次观看.  Eastern Virginia Medical School. We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish. 

视频: A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

配备了光学切片与成像定时使用解剖显微镜斑马鱼肾脏发展分析

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

科研

发育生物学

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

精确的细胞消融方法建模发展中的斑马鱼急性肾损伤

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be difficult to study, and can lead to confounding results. The protocol described here is a straightforward breeding paradigm to increase and decrease penetrance in lethal zebrafish skeletal mutants. Because lethal mutants cannot be selectively bred directly, the classic selective breeding strategy of progeny testing is employed. This method also includes protocols for Kompetitive Allele Specific PCR (KASP) genotyping zebrafish and staining larval zebrafish cartilage and bone. Applying the husbandry strategy described here can increase the penetrance of an interesting skeletal phenotype enabling more reproducible results in downstream applications. In addition, decreasing the mutant penetrance through this selective breeding strategy can reveal the developmental processes that most crucially require the function of the mutated gene. While the skeleton is specifically considered here, we propose that this methodology will be useful for all zebrafish mutant lines.

The goal of this protocol is to alter the penetrance of lethal skeletal mutant phenotypes in zebrafish by selective breeding. Lethal mutants cannot be grown to adulthood and bred themselves, therefore this protocol describes a method for tracking and selecting penetrance through multiple generations by progeny testing.

通过子代检测改变斑马鱼致死性骨骼突变显

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be ...

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

全贴装荧光原位杂交和免疫荧光法在斑马鱼中 Multiciliated 细胞的可视化

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Behavioral And Physiological Analysis In A Zebrafish Model Of Epilepsy

Epilepsy represents one of the most common neurological disorders, affecting an estimated 50 million people worldwide. Recent advances in genetic research have uncovered a large spectrum of genes implicated in various forms of epilepsy, highlighting the heterogeneous nature of this disorder. Appropriate animal models are essential for investigating the pathological mechanisms triggered by genetic mutations implicated in epilepsy and for developing specialized, targeted therapies. In recent years, zebrafish has emerged as a valuable vertebrate organism for modeling epilepsies, with the use of both genetic manipulation and exposure to known epileptogenic drugs, such as pentylenetetrazole (PTZ), to identify novel anti-epileptic therapeutics. Deleterious mutations in the mTOR regulator DEPDC5 have been associated with various forms of focal epilepsies and knock-down of the zebrafish orthologue causes hyperactivity associated with spontaneous seizure-like episodes, as well as enhanced electrographic activity and characteristic turn wheel swimming. Here, we described the method involved in generating the DEPDC5 loss-of-function model and illustrate the protocol for assessing motor activity at 28 and 48 h post fertilization (hpf), as well as a method for recording field activity in the zebrafish optic tectum. An illustration of the effect of the epileptogenic drug PTZ on neuronal activity over time is also provided.

Here, we present a protocol for the development and the characterization of a zebrafish model of epilepsy resulting from the transient inhibition of the DEPDC5 gene.

斑马鱼癫痫模型中的行为和生理分析

Epilepsy represents one of the most common neurological disorders, affecting an estimated 50 million people worldwide. Recent advances in genetic research ...

神经科学

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

在体内病态的人类基因组建模使用斑马鱼

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

生物学

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

颅面部发育中的SOX10可视化：使用枫转基因斑马鱼线时间推移共聚焦显微镜

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

肾单位的组成与功能的成年斑马鱼肾脏分析

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...

Acute Kidney Injury Model Induced by Cisplatin in Adult Zebrafish

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney due to its low molecular weight. Such accumulation causes the death of tubular cells and can induce the development of Acute Kidney Injury (AKI), which is characterized by a quick decrease in kidney function, tissue damage, and immune cells infiltration. If administered in specific doses cisplatin can be a useful tool as an AKI inducer in animal models. The zebrafish has appeared as an interesting model to study renal function, kidney regeneration, and injury, as renal structures conserve functional similarities with mammals. Adult zebrafish injected with cisplatin shows decreased survival, kidney cell death, and increased inflammation markers after 24 h post-injection (hpi). In this model, immune cells infiltration and cell death can be assessed by flow cytometry and TUNEL assay. This protocol describes the procedures to induce AKI in adult zebrafish by intraperitoneal cisplatin injection and subsequently demonstrates how to collect the renal tissue for flow cytometry processing and cell death TUNEL assay. These techniques will be useful to understand the effects of cisplatin as a nephrotoxic agent and will contribute to the expansion of AKI models in adult zebrafish. This model can also be used to study kidney regeneration, in the search for compounds that treat or prevent kidney damage and to study inflammation in AKI. Moreover, the methods used in this protocol will improve the characterization of tissue damage and inflammation, which are therapeutic targets in kidney-associated comorbidities.

This protocol describes the procedures to induce Acute Kidney Injury (AKI) in adult zebrafish using cisplatin as a nephrotoxic agent. We detailed the steps to evaluate the reproducibility of the technique and two techniques to analyze inflammation and cell death in the renal tissue, flow cytometry and TUNEL, respectively.

成人斑马鱼中西斯铂诱导的急性肾损伤模型

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney ...

医学

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish kidney develops and functions in almost the same way as humans. A major difference in the construction of the human kidney is the presence of millions of nephrons compared to the zebrafish that has only two. However, simplifying such a complex system into basic functional units has aided our understanding of how the kidney develops and operates. In zebrafish, the midline located glomerulus is responsible for the initial blood filtration into two pronephric tubules that diverge to run bilaterally down the embryonic axis before fusing to each other at the cloaca. The pronephric tubules are heavily populated by motile cilia that facilitate the movement of filtrate along the segmented tubule, allowing the exchange of various solutes before finally exiting via the cloaca2-4. Many genes responsible for CKD, including those related to ciliogenesis, have been studied in zebrafish5. However, a major draw back has been the difficulty in evaluating zebrafish kidney function after genetic manipulation. Traditional assays to measure kidney dysfunction in humans have proved non translational to zebrafish, mainly due to their aquatic environment and small size. For example, it is not physically possible to extract blood from embryonic staged fish for analysis of urea and creatinine content, as they are too small. In addition, zebrafish do not produce enough urine for testing on a simple proteinuria &#8216;dipstick&#8217;, which is often performed during initial patient examinations. We describe a fluorescent assay that utilizes the optical transparency of the zebrafish to quantitatively monitor the clearance of a fluorescent dye, over time, from the vasculature and out through the kidney, to give a read out of renal function1,6-9.

The zebrafish is a popular tool to model chronic kidney disease (CKD). However, their small size makes it impossible to evaluate renal function using traditional methods. We describe a fluorescent dye kidney clearance assay1 that allows quantitative analysis of zebrafish kidney function in CKD.

斑马鱼肾功能评价采用荧光测定间隙

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish ...

多囊肾一种可能的斑马鱼型号:击倒

摘要

探索更多视频

此视频中的章节